The goal of this SBIR Phase I proposal is i) to develop a commercial prototype NanoVelcro Assay that uses thermoresponsive polymers for circulating tumor cell (CTC) capture and release and ii) to validate the clinical feasibility of performing a streamlined process starting from CTC capture, isolationrelease, and finishing with recovery of viable CTCs for molecular and functional analysis of cancer patient blood. This proposal is led by Dr. Garcia (PI), who has extensive experience in early-stage development of the TR-NanoVelcro CTC Assay and has a background in nanomaterials, surface chemistry, microfluidics, and in vitro diagnostic technologies. CTCs are cancer cells shed from either the primary tumors or metastatic sites, and throughout the malignant transformation process, they travel through the blood stream to different tissues, presaging or perpetuating metastases. However, CTC detection and characterization have been technically challenging due to extremely low CTC abundance (~1-100s CTCsmL) among hematologic cells. Currently, CellSearchTM Assay is the only FDA-cleared device, but it is costly and inefficient in capturing CTCs, and it is not amenable for downstream molecular or functional analysis. Thus, there is a need for a commercial CTC assay that goes beyond simple enumeration - one which is inexpensive, able to recover viable CTCs for molecular and functional analysis, and can be easily integrated into a clinical setting. Under sequential funding support (via R21 and R33) from the NCI IMAT program, CytoLumina's academic consultants at UCLA and Cedars-Sinai demonstrated a highly efficient, inexpensive cell-affinity assay capable of enriching, identifying, and isolating CTCs from cancer patient blood. The team pioneered the development of NanoVelcro substrates, in which a capture agent is coated onto a nanostructured surface in order to attract CTCs in a stationary device setting. In order to enable greater blood volume throughput, thereby increasing the number of CTCs captured, the team positioned a polydimethylsiloxane chaotic mixer over the substrate to induce vertical flow of the blood over the nanostructured surface. First, CytoLumina will spike 100 patient derived prostate cancer cells into 2 mL of healthy-donor blood to generate an artificial CTC sample that will be used to determine capture efficiency and recovery performance. After optimization, the clinical feasibility will be assessed by collecting 10 prostate patient blood samples and running them through the TR-NanoVelcro Assay. Finally, we will generate RT-PCR data on 14 genes and obtain two patient-CTC derived primary cell lines to demonstrate the diagnostic and clinical value of the TR-NanoVelcro CTC Assay. The successful demonstration of this proposal will enable a wide range of applications, for example, generating patient CTC-derived cell lines and using CTCs as an in vitro screening approach of potential therapeutics for personalized medicine.